The present invention concerns a process and a reagent for the determination of .alpha.-amylase by enzymatic splitting of a substituted nitrophenylated maltoheptaoside, in which the aglycone is .beta.-glycosidically bound, in the presence of .alpha.-glucosidase and .beta.-glucosidase and measurement of the liberated aglycone.
The determination of the .alpha.-amylase activity in body fluids, e.g. serum, plasma, urine, duodenal juice, saliva, has a great importance in the clinical-chemical diagnosis for the recognition of pancreatic diseases.
The previously known methods for the determination of .alpha.-amylase depend upon the fact that starch is broken down by .alpha.-amylase and the fragments formed are determined spectrophotometrically. The important disadvantage of these methods is that starch, as the macromolecule, is difficult to characterize and to standardize.
Therefore, more recent methods use defined oligosaccharides as substrates. These are broken down in the presence of .alpha.-glucosidase as far as glucose, which is determined in per se known manner. (See, e.g. U.S. Pat. No. 4,000,042 and U.S. Pat. No. 3,879,263.) However, these reactions are very complex and are disturbed by endogenic glucose. A considerable simplification was achieved by the use of 4-nitrophenylated oligosaccharides with a chain length of 3 to 12 glucose units (G3-G12) as substrate for the .alpha.-amylase. By means of a subsequent .alpha.-glucosidase reaction, the liberated p-nitrophenol is measured (published Federal Republic of Germany Patent Application No. 27 31 421 and U.S. Pat. No. 4,233,403). In U.S. Pat. No. 4,145,527, there are described substrates for .alpha.-amylase with up to 6 glucose units, in the case of the production of which a mixture of the .alpha.- and .beta.-isomers is always obtained, which is used together with .alpha.- and .beta.-glucosidases. However, from Fresenius Z. Anal. Chem., 301, 169 (1980) it is known that with the .beta.-isomers, in contradistinction to the .alpha.-isomers, substantially smaller activities are always measured. In addition, U.S. Pat. No. 4,102,747 discloses the determination of .alpha.-amylase using a mixture of .alpha.- and .beta.-p-nitrophenyl isomers of G4-G10 oligosaccharides in combination with both .alpha.- and .beta.-glucosidases. Similarly, DOS No. 2755803 discloses the use of G7 oligosaccharides having .beta.-aglycones, including nitro- and dinitrophenyl, in combination with .alpha.- and .beta.-glucosidases.
If, in the prior art, the p-nitrophenol is preferably used as the measured variable, then additional disadvantages arise: the pKa value of p-nitrophenol lies at 7.09; this means that, under optimum pH conditions for the course of the .alpha.-amylase determination (6.8-7.1), only about 50% of the liberated p-nitrophenol is present as colored phenolate anion and the resulting measurement signal is correspondingly low. Furthermore, changes in the hydrogen ion concentration of the test system, perhaps due to the use of acidic or alkaline samples, such as urine or duodenal juice, can change the degree of dissociation of the liberated p-nitrophenol and thus the apparent molar extinction coefficients.